title: The removal of a disulfide bridge in CotA-laccase changes the slower motion dynamics involved in copper binding but has no effect on the thermodynamic stability.
abstract: The contribution of the disulfide bridge in CotA-laccase from Bacillus subtilis is assessed with respect to the enzyme's functional and structural properties. The removal of the disulfide bond by site-directed mutagenesis, creating the C322A mutant, does not affect the spectroscopic or catalytic properties and, surprisingly, neither the long-term nor the thermodynamic stability parameters of the enzyme. Furthermore, the crystal structure of the C322A mutant indicates that the overall structure is essentially the same as that of the wild type, with only slight alterations evident in the immediate proximity of the mutation. In the mutant enzyme, the loop containing the C322 residue becomes less ordered, suggesting perturbations to the substrate binding pocket. Despite the wild type and the C322A mutant showing similar thermodynamic stability in equilibrium, the holo or apo forms of the mutant unfold at faster rates than the wild-type enzyme. The picosecond to nanosecond time range dynamics of the mutant enzyme was not affected as shown by acrylamide collisional fluorescence quenching analysis. Interestingly, copper uptake or copper release as measured by the stopped-flow technique also occurs more rapidly in the C322A mutant than in the wild-type enzyme. Overall the structural and kinetic data presented here suggest that the disulfide bridge in CotA-laccase contributes to the conformational dynamics of the protein on the microsecond to millisecond timescale, with implications for the rates of copper incorporation into and release from the catalytic centres.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Oeiras, Portugal.
authors.forename: André T
authors.initials: AT
authors.lastname: Fernandes
authors.forename: Manuela M
authors.initials: MM
authors.lastname: Pereira
authors.forename: Catarina S
authors.initials: CS
authors.lastname: Silva
authors.forename: Peter F
authors.initials: PF
authors.lastname: Lindley
authors.forename: Isabel
authors.initials: I
authors.lastname: Bento
authors.forename: Eduardo Pinho
authors.initials: EP
authors.lastname: Melo
authors.forename: Lígia O
authors.initials: LO
authors.lastname: Martins
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D055162:Biocatalysis; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D055550:Protein Stability; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D003300:Copper; D042845:Laccase